Fusion proteins of collagen-binding domain and parathyroid hormone

ABSTRACT

Fusion proteins containing active agonist or antagonist fragments of parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP) coupled to a collagen-binding domain are presented. The fusion proteins can be used to promote bone growth, to promote hair growth, to prevent cancer metastasis to bone, to promote immune reconstitution with a bone marrow stem cell transplant, to promote mobilization of bone marrow stem cells for collection for autologous stem cell transplant, and to treat renal osteodystrophy. Pharmaceutical agents comprising a collagen-binding polypeptide segment linked to a non-peptidyl PTH/PTHrP receptor agonist or antagonist are also presented.

BACKGROUND

Osteoporosis is a bone disease characterized by thinning of bone tissueand loss of bone density over time. It is widely prevalent in theelderly. The National Osteoporosis Foundation estimates that by 2020nearly 14 million Americans will suffer from osteoporosis. An additional18 million may have low bone mass, or osteopenia. Osteoporosis can occureither because the body fails to make enough new bone or reabsorbs toomuch old bone, or both.

Osteoporosis often progresses painlessly until a bone breaks. Any bonecan be affected, but one of principal concern is the hip. A hip fractureimpairs a person's ability to walk and causes prolonged and sometimespermanent disability.

Osteoporosis can be treated with anabolic therapies or antiresorptivetherapies. Anabolic therapies build new bone. But antiresporptivetherapies do not. Instead they slow the resorption of existing bone. Amajor factor in the control of bone remodeling is parathyroid hormone(PTH). PTH and its analogs are the only class of anabolic therapeuticswith proven clinical efficacy. Teriparatide is an approved therapeuticthat is a shortened version of PTH. It consists of the N-terminal 34amino acid residues of mature PTH (PTH(1-34)). Teriparatide isadministered by once daily subcutaneous injection.

PTH is an 84-amino acid peptide. It is involved in mineral ionhomeostasis. Increased PTH mobilizes calcium from bone in response tocalcium deficient diets or vitamin D insufficiency. PTH also affectsosteoblasts and stromal cells. Although hyperparathyroidism isassociated with bone loss, PTH administration causes bone gain. PTHbinds to receptors on osteoblasts, specialized bone cells thatsynthesize bone, and this appears to prolong osteoblast life andincrease osteoblast activity, causing bone gain.

PTH-related peptide (PTHrP) is a 141-amino acid protein that ishomologous to PTH over its first 13 amino acids but diverges thereafter(1-3). PTH and PTHrP act through a common PTH/PTHrP receptor.

New treatments for osteoporosis are needed. Improved methods to deliverPTH, teriparatide, or other PTH/PTHrP receptor agonist agents areneeded.

SUMMARY

One embodiment disclosed herein involves compositions or bioactiveagents comprising a collagen-binding polypeptide segment linked to aPTH/PTHrP receptor agonist. The inventors have constructed fusionproteins containing residues 1-33 of PTH, an active agonist fragment ofPTH, fused to a collagen-binding domain (CBD) of ColH, a collagenasefrom Clostridium histolyticum. The inventors have found that the fusionprotein is more active than PTH(1-34) in promoting bone growth in vivoin mice, even when administered systemically. With local administrationto, for instance, a fracture site, the difference in efficacy isexpected to be even greater. Peptides that are antagonists of thePTH/PTHrP receptor can also be coupled to a CBD for targeted andenhanced bioactivity.

Compositions or bioactive agents containing a collagen-bindingpolypeptide segment coupled to a non-peptidyl agonist or antagonist ofthe PTH/PTHrP receptor are also presented.

Collagen is the most abundant protein in mammals. It is the majorprotein component of bone and cartilage. A CBD-bioactive agent fusionprotein thus targets the bioactive agent to collagen, and generally tobone and cartilage. The CBD-PTH fusion proteins have longer half-livesthan PTH because of their stable binding to collagen, which tends toremove them from circulation. They can be administered locally, forinstance, at a fracture site, and will tend to remain at the site ofadministration through binding to collagen at or near the site ofadministration. In support of this longer half-life, a fusion proteincontaining epidermal growth factor (EGF) with a CBD was shown to havemuch longer half life than EGF alone (8). Data is also presented inExamples 4 and 5 herein showing that a PTH-CBD fusion proteinadministered weekly or monthly is as effective or more effective thanPTH(1-34) administered daily.

One embodiment provides a composition comprising: a collagen-bindingpolypeptide segment linked to a PTH/PTHrP receptor agonist; wherein thecollagen-binding polypeptide segment is a bacterial collagen-bindingpolypeptide segment.

One embodiment provides a composition comprising: a collagen-bindingpolypeptide segment linked to a PTH/PTHrP receptor agonist; wherein thecollagen-binding polypeptide segment is a segment of a collagenase.

One embodiment provides a composition comprising: a collagen-bindingpolypeptide segment linked to a PTH/PTHrP receptor agonist; wherein,over an 8-week period, the increase in bone mineral density of thecomposition injected with a vehicle intraperitoneally weekly in a mouserelative to the vehicle alone is at least 50% larger than the increasein bone mineral density of an equimolar amount of a compositionconsisting of the PTH/PTHrP agonist relative to the vehicle alone.

That is, the bioactive agent (composition) causes an increase in bonemineral density in mice when administered at an appropriate dose in avehicle, such as an aqueous buffer solution. A control treatment withthe vehicle alone may also result in some change in bone mineraldensity, for example because the mice are juveniles that are stillgrowing or elderly mice whose bone mineral density is otherwisedeclining. The appropriate way to measure the effect of the bioactiveagent is to measure increase in bone mineral density in experimentalmice treated with the agent minus increase (or decrease) in bone mineraldensity in control mice treated with vehicle alone. This increase inbone mineral density with administration of the agent after correctionfor change in bone mineral density in control mice receiving vehiclealone is at least 50% larger than the increase in bone mineral densityin mice treated with an agent containing only the PTH/PTHrP receptoragonist (not coupled to a collagen-binding polypeptide segment), againafter correcting for any changes in bone mineral density in control micetreated with vehicle alone. For instance, in FIG. 3 herein, described inExample 4, the vehicle control mice have an increase in bone mineraldensity during an 8-week treatment period of 5%, mice treated withPTH(1-34) (a PTH/PTHrP agonist) have an increase in BMD of about 7.5%,and mice treated with a PTH-CBD fusion protein containing PTH(1-33)coupled to a collagen-binding domain have an increase in BMD of over15%. The mice treated with the PTH-CBD fusion protein thus have anincrease in BMD after correcting for the change with vehicle alone ofover 10% (over 15% minus 5%), and the mice treated with PTH(1-34) havean increase in BMD after correcting for the change with vehicle alone ofabout 2.5% (about 7.5% minus 5%). Thus, intraperitoneal weekly injectionof the fusion protein causes over 300% more (over 4-times as much, over10% versus about 2.5%) increase in BMD as injection of the PTH(1-34).

Another embodiment provides a fusion protein comprising: abacterialcollagen-binding polypeptide segment; linked to a PTH/PTHrP receptoragonist polypeptide segment.

Another embodiment provides a fusion protein comprising: acollagen-binding polypeptide segment of a collagenase; linked to aPTH/PTHrP receptor agonist polypeptide segment.

Another embodiment provides a fusion protein comprising: acollagen-binding polypeptide segment; linked to a PTH/PTHrP receptorantagonist polypeptide segment.

Another embodiment provides a composition comprising: a collagen-bindingpolypeptide segment; linked to a non-peptidyl PTH/PTHrP receptoragonist.

Another embodiment provides a composition comprising: a collagen-bindingpolypeptide segment; linked to a non-peptidyl PTH/PTHrP receptorantagonist.

Another embodiment provides a composition comprising: a collagen-bindingpolypeptide segment; linked to a PTH/PTHrP receptor antagonist.

Another embodiment provides a method of promoting bone growth in amammal comprising: administering to the mammal a composition comprising:(a) a collagen-binding polypeptide segment; linked to (b) a PTH/PTHrPreceptor agonist.

Another embodiment provides a method of promoting bone growth in amammal comprising: administering to the mammal a composition comprising(a) a collagen-binding polypeptide segment; linked to (b) a PTH/PTHrPreceptor agonist.

Another embodiment provides a method of promoting hair growth in amammal comprising: administering to the mammal a composition comprising:(i) a collagen-binding polypeptide segment; linked to (ii) a PTH/PTHrPreceptor agonist polypeptide segment.

Another embodiment provides a method of promoting hair growth in amammal comprising: administering to the mammal a composition comprising:(i) a collagen-binding polypeptide segment; linked to (ii) a PTH/PTHrPreceptor antagonist.

Another embodiment provides a method of promoting tissue growth aroundan implant in a mammal comprising: administering to the mammal acomposition comprising (a) a collagen-binding polypeptide segment;linked to (b) a PTH/PTHrP receptor agonist; wherein before, during, orafter the step of administering the composition, the mammal receives animplant placed in contact with tissue in the mammal; and wherein thestep of administering the composition is effective to promote tissuegrowth around the implant.

Another embodiment provides a method of promoting immune reconstitutionin a mammal comprising: administering to the mammal a compositioncomprising: (a) a collagen-binding polypeptide segment; linked to (b) aPTH/PTHrP receptor agonist; wherein before, during, or after the step ofadministering the composition, the mammal receives an administration ofbone marrow stem cells. The composition enhances immune reconstitutionby enhancing grafting, multiplication, and/or differentiation of thebone marrow stem cells.

Another embodiment provides a method of promoting bone marrow stem cellmobilization in a mammal comprising: administering to the mammal acomposition comprising: (a) a collagen-binding polypeptide segment;linked to (b) a PTH/PTHrP receptor agonist; wherein administering thecomposition increases the number of stem cells in circulating blood ofthe mammal (e.g., 7, 14, or 30 days after administering the fusionprotein).

Another embodiment provides a method of treating or preventing renalosteodystrophy in a mammal comprising: administering to the mammal acomposition comprising: (a) a collagen-binding polypeptide segment;linked to (b) a PTH/PTHrP receptor antagonist; wherein the mammal isafflicted with renal osteodystrophy or renal disease and the compositionis effective to reduce bone loss in the mammal.

Another embodiment provides a method of treating or preventing (i.e.,reducing incidence of) bone metastasis of cancer in a mammal comprising:administering to the mammal a composition comprising: (a) acollagen-binding polypeptide segment; linked to (b) a PTH/PTHrP receptorantagonist; wherein the composition is administered at a dosageeffective to reduce incidence of bone metastasis of cancer or slow thegrowth of metastatic cancer in bone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an SDS-PAGE gel showing the results of an experiment showingthat two PTH-CBD fusion proteins bind to collagen.

FIG. 2 is a graph showing in vitro cAMP accumulation in cells stimulatedwith PTH(1-34) or PTH-CBD fusion proteins.

FIG. 3 is a bar graph showing increase in spinal bone mineral density inmice treated with weekly intraperitoneal injection for 8 weeks of buffer(vehicle), PTH(1-34), PTH-PKD-CBD fusion protein, or PTH-CBD fusionprotein.

FIG. 4 is a bar graph showing absolute spinal bone mineral density ofexcised spine segments from mice sacrificed after treatment for 8 weekswith weekly intraperitoneal injection of buffer (vehicle), PTH(1-34),PTH-PKD-CBD fusion protein, or PTH-CBD fusion protein.

FIG. 5 is a bar graph showing serum calcium levels of mice after 8 weeksof weekly injections of buffer (vehicle), PTH(1-34), PTH-PKD-CBD fusionprotein, or PTH-CBD fusion protein.

FIG. 6 is a bar graph showing serum alkaline phosphatase concentrationof mice after 8 weeks of weekly injections of buffer (vehicle),PTH(1-34), PTH-PKD-CBD fusion protein, or PTH-CBD fusion protein.

FIG. 7 is a micrograph of sections of tibia bone from a vehicle-treatedcontrol mouse and a mouse receiving 8 weeks of weekly injection ofPTH-CBD fusion protein. The sections were stained with hematoxylin andeosin stain. The micrograph shows increased cortical and trabecular bonemass in the bone of the mouse treated with PTH-CBD.

FIG. 8 is line graph of bone mineral density over time for mice treatedmonthly with PTH-CBD, PTH(1-34), or vehicle control for 6 months. At 6months, the group receiving PTH(1-34) was treated daily for two weeks(indicated by the arrow on the X axis). Then all groups were untreatedfor the rest of the study.

FIG. 9 is a line graph of bone mineral density over time for micetreated with PTH(1-34) daily for 14 days (PTH), with the PTH-CBD fusionprotein once at the initiation of the study (CBD-PTH-6), with PTH-CBDfusion protein at time 0 and a second time at 3 months (CBD-PTH-3), andwith vehicle control.

FIG. 10 is a bar graph showing serum alkaline phosphatase concentrationof mice after 8 weeks of weekly injections of buffer (vehicle),PTH(1-34), PTH-PKD-CBD fusion protein, or PTH-CBD fusion protein.

FIG. 11 is a bar graph of bone mineral density in mice receiving asingle dose of a range of dosage amounts of PTH-CBD by subcutaneousinjection. Bone mineral density was followed for 32 weeks. Each dosagewas given to two mice.

FIG. 12 shows photographs of mice described in Example 8 havingchemotherapy-induce alopecia and a shaved spot on their backs, treatedwith the PTH-CBD fusion protein by subcutaneous injection at thehairless spot, or untreated controls. There are 3 mice in each group,and photos are taken at 0 days, 14 days, and 21 days after the injectionof PTH-CBD. The photos show greater hair growth in the subjects treatedwith the PTH-CBD fusion protein.

DETAILED DESCRIPTION

This disclosure involves compositions, including bioactive agents andfusion proteins, comprising a collagen-binding polypeptide segmentlinked to a PTH/PTHrP receptor agonist or antagonist. In a preferredembodiment, the compositions are fusion proteins where the PTH/PTHrPagonist or antagonist is a polypeptide segment, where thecollagen-binding polypeptide segment and PTH/PTHrP polypeptide segmentare linked together in a fusion protein. But the PTH/PTHrP agonist orantagonist portion can also be a non-peptidyl agonist or antagonist.

The terms “fusion protein” and “fusion polypeptide” may be used to referto a single polypeptide comprising two functional segments, e.g., acollagen-binding polypeptide segment and a PTH/PTHrP receptor agonistpolypeptide segment. The fusion proteins may be any size, and the singlepolypeptide of the fusion protein may exist in a multimeric form in itsfunctional state, e.g., by cysteine disulfide connection of two monomersof the single polypeptide. A polypeptide segment may be a syntheticpolypeptide or a naturally occurring polypeptide. Such polypeptides maybe a portion of a polypeptide or may comprise a mutation.

The collagen-binding polypeptide segment is a polypeptide that bindscollagen and may be part of a larger fusion protein, bioactive agent, orpharmaceutical agent. Determination of whether a composition,polypeptide segment, fusion protein, or pharmaceutical or bioactiveagent binds collagen can be made as described in Example 2 below.Briefly, it is incubated with collagen in binding buffer, and themixture is then filtered through a filter that would otherwise allow itto pass through but that blocks the collagen and therefore holds backmaterials that bind to the collagen. The filtrate is then assayed forthe presence of the composition, polypeptide segment, fusion protein, orpharmaceutical or bioactive agent. Preferably, at least 90%, morepreferably at least 99% of the collagen-binding composition, polypeptidesegment, fusion protein, or pharmaceutical or bioactive agent isretained by the filter in this assay, as compared to when the filtrationis performed without collagen.

One embodiment disclosed herein involves fusion proteins comprising acollagen-binding polypeptide segment linked to a PTH/PTHrP receptoragonist polypeptide segment.

The PTH/PTHrP receptor agonist polypeptide segment may be a syntheticpolypeptide or a naturally occurring polypeptide. Such polypeptides maybe a portion of a polypeptide or may comprise a mutation. Agonistactivity with the PTH/PTHrP receptor can be assayed as described inExample 3 below by a cAMP stimulation assay. An agonist will stimulatecAMP synthesis. Preferably, an agonist can activate receptor activity atleast 10% as much as PTH(1-34).

In a specific embodiment when injected intraperitoneally weekly in micethe agonist fusion protein causes at least 50% more increase in bonemineral density (as compared to vehicle control) than an equimolaramount of a polypeptide consisting of the PTH/PTHrP receptor agonistpolypeptide segment when injected intraperitoneally weekly (as comparedto vehicle control) over an 8-week period (as in Example 4 below).Likewise, in other specific embodiments, the fusion protein causes astatistically significantly (p<0.05) greater increase in BMD, or atleast twice as much increase in BMD, than an equimolar amount of apolypeptide consisting of the PTH/PTHrP receptor agonist polypeptidesegment or than PTH(1-34).

In some embodiments of the fusion proteins, the collagen-bindingpolypeptide segment is a bacterial collagen-binding polypeptide segment.In a more specific embodiment, it is a Clostridium collagen-bindingpolypeptide segment.

In some embodiments of the fusion proteins, the collagen-bindingpolypeptide segment is a segment of a collagenase, or a bacterialcollagenase, or a Clostridium collagenase. Preferably the segment isonly a portion of the collagenase and the collagen-binding polypeptidesegment does not have collagenase activity.

In some embodiments, the collagenase is ColH, SEQ ID NO:6.

In some embodiments, the collagen-binding polypeptide segment is orincludes residues 901-1021 of SEQ ID NO:6 (residues 38-158 of SEQ IDNO:1), or a fragment of residues 38-158 of SEQ ID NO:1 at least 8 aminoacid residues in length.

In some embodiments, the collagen-binding polypeptide segment is atleast 90%, at least 95%, at least 96%, at least 98%, or at least 99%identical to residues 38-158 of SEQ ID NO:1.

In some embodiments, the collagen-binding polypeptide segment is orincludes residues 807-1021 of SEQ ID NO:6 (residues 37-251 of SEQ IDNO:2).

In specific embodiments, the collagen-binding polypeptide segment is orcomprises a fragment of residues 901-1021 of SEQ ID NO:6, e.g., afragment of at least 8, at least 10, at least 20, at least 30 at least40, or at least 50 consecutive amino acid residues of residues 901-1021of SEQ ID NO:6.

Among other proteins the collagen-binding segment can be derived fromare ColG (5), a class I collagenase from Clostridium histolyticum. ColHis a class II collagenase (6).

The collagen-binding polypeptide segment may also be a polypeptidesegment from bone sialoprotein, fibronectin, or von Willebrand factor,as described in references (30-33), or may be polyglutamic acid (34).

In specific embodiments, the PTH/PTHrP receptor agonist polypeptidesegment is a PTH or PTHrP polypeptide segment One human isoform of PTHis SEQ ID NO:7. One human isoform of PTHrP is SEQ ID NO:8.

In specific embodiments, the PTH/PTHrP receptor agonist polypeptidesegment is or includes residues 1-33 of SEQ ID NO:1 (residues 1-33 ofPTH (SEQ ID NO:7)).

In specific embodiments, the PTH/PTHrP receptor agonist polypeptidesegment is or includes residues 1-34 of PTH (SEQ ID NO:7). In otherembodiments, it is a fragment of residues 1-34 of PTH (SEQ ID NO:7).

In specific embodiments, the PTH/PTHrP receptor agonist polypeptidesegment is or includes residues 1-84 of PTH (SEQ ID NO:7).

In specific embodiments, the PTH/PTHrP receptor agonist polypeptidesegment is or includes residues 1-14 of PTH (SEQ ID NO:7).

In specific embodiments, the PTH/PTHrP receptor agonist is a PTH orPTHrP polypeptide segment.

In one embodiment, the PTH/PTHrP receptor agonist polypeptide segment isN terminal to the collagen-binding polypeptide segment in the fusionprotein. That is, the two polypeptide segments each have an N-terminaland a C-terminal, and the N-terminal of the collagen-binding polypeptidesegment is linked directly or through a linker polypeptide segment tothe C-terminal of the PTH/PTHrP agonist polypeptide segment.

The two polypeptide segments of the fusion proteins can be linkeddirectly or indirectly. For instance, the two segments may be linkeddirectly through, e.g., a peptide bond or chemical cross-linking, orindirectly, through, e.g., a linker segment or linker polypeptide.

This disclosure also provides a fusion protein comprising acollagen-binding polypeptide segment linked to a PTH/PTHrP receptorantagonist polypeptide segment.

The PTH/PTHrP receptor antagonist polypeptide segment may be a syntheticpolypeptide or a naturally occurring polypeptide. Such polypeptides maybe a portion of a polypeptide or may comprise a mutation. Antagonistactivity with the PTH/PTHrP receptor can be assayed as described inExample 3 below by a cAMP stimulation assay. An antagonist will inhibitstimulation of cAMP synthesis by PTH(1-34). Preferably, when mixed withPTH(1-34), the antagonist can inhibit activation of the receptor byPTH(1-34) by at least 50%. In contrast, when not mixed with PTH, theantagonist activates the receptor by less than 5% of the receptor'smaximal activation by PTH(1-34).

In the fusion proteins containing a PTH/PTHrP receptor antagonist, thecollagen-binding polypeptide segment can be the same segments as foundin the fusions containing a PTH/PTHrP receptor agonist.

In some embodiments, the PTH/PTHrP receptor antagonist is a PTH or PTHrPpolypeptide segment.

The PTH/PTHrP receptor antagonist can include in one embodimentPTH(7-34), i.e., residues 7-34 of PTH (SEQ ID NO:7). In anotherembodiment, it is or includes residues 7-33 of PTH (SEQ ID NO:7). Inother embodiments, it is a fragment of residues 7-34 of SEQ ID NO:8.

In another embodiment, the PTH/PTHrP receptor antagonist includesPTH(7-14), i.e., residues 7-14 of PTH (SEQ ID NO:7).

In another embodiment, the PTH/PTHrP receptor antagonists includeresidues 1-14 of PTH with an N-terminal extension. Adding an N-terminalextension to PTH or active N-terminal fragments of PTH converts the PTHpeptides to antagonists. The N-terminal extension can be 1, 2, 3, 4, 5,or more amino acids in length. The identity of the amino acids in theN-terminal extension is typically not important. In one embodiment, thePTH/PTHrP receptor antagonist includes residues 1-33 of PTH with aGly-Ser extension at the N-terminus (SEQ ID NO:11).

In another embodiment, the PTH/PTHrP receptor antagonist includesPTHrP(7-34), i.e., residues 7-34 of SEQ ID NO:8, or a fragment ofresidues 7-34 of SEQ ID NO:8.

In another embodiment, the PTH/PTHrP receptor antagonist includes mouseTIP(7-39) (reference 18). Other PTH/PTHrP receptor antagonists that maybe used in the fusion proteins are also disclosed in reference (18).

In one embodiment, the PTH/PTHrP receptor antagonist polypeptide segmentis N terminal to the collagen-binding polypeptide segment in theantagonist fusion protein. That is, the two polypeptide segments eachhave an N-terminal and a C-terminal, and the N-terminal of thecollagen-binding polypeptide segment is linked directly or through alinker polypeptide segment to the C-terminal of the PTH/PTHrP antagonistpolypeptide segment.

As with the agonist, the two polypeptide segments of the antagonistfusion proteins can be linked directly or indirectly.

This disclosure also provides a method of promoting bone growth in amammal involving administering to the mammal a fusion protein comprisinga collagen-binding polypeptide segment linked to a PTH/PTHrP agonistpolypeptide segment.

In particular embodiments, administering the fusion protein to themammal increases trabecular bone mineral volume and/or trabecular bonemineral density or slows loss of trabecular bone mineral volume and/ortrabecular bone mineral density.

In particular embodiments, administering the fusion protein to themammal increases cortical bone mineral volume and/or cortical bonemineral density or slows loss of cortical bone mineral volume and/orcortical bone mineral density.

Bone mineral volume is visible from histologic staining of slides. Theterm “bone mineral volume” as used herein refers to the volume occupiedby mineralized bone. “Bone mineral density” as used herein refers toareal bone density, i.e., the amount of bone mineral per unit2-dimensional area of bone. It can be measured by x-rays, or DEXA(Example 4 below).

The inventors have found that the PTH-CBD fusion protein increases boththe bone mineral volume and density of both trabecular and corticalbone. The effect on cortical bone is surprising, because PTH(1-34) hasbeen shown to have little effect on cortical bone mineral density oreven decrease cortical bone mineral density, even as it increasestrabecular bone mineral density (25-27).

The fusion protein can be administered systemically, e.g., byintravenous injection. The inventors have found that when administeringthe fusion protein subcutaneously it binds locally at the site ofinjection if the fusion protein is dissolved in neutral pH buffer. Butif the fusion protein is dissolved in pH 4.5 or below buffer, thecollagen-binding domain does not bind collagen, and the fusion proteinhas time to disperse systemically before it binds collagen elsewhere inthe body at neutral pH. Thus, in one embodiment, systemic administrationof the fusion proteins involves administering the fusion proteindissolved in buffer or aqueous solution at a pH lower than about 5.0 orat pH 4.5 or below. In another embodiment, systemic administration ofthe fusion proteins involves administering the fusion proteins dissolvedin aqueous solution at pH lower than about 6.0.

In particular embodiments, the fusion protein is administered byinjection, e.g., intravenous or subcutaneous or intraperitonealinjection. Administration by injection may be systemic administration orlocal administration.

In particular embodiments, the fusion protein is administered in anorthopedic implant. Examples of orthopedic implants in which the fusionprotein may be administered include an orthopedic bone void filler, anadjunct to bone fracture stabilization, an intramedullary fixationdevice, a joint augmentation/replacement device, a bone fixation plate,a screw, a tack, a clip, a staple, a nail, a pin, a rod, an anchor, ascrew augmentation device, or a cranial reconstruction device. Anotherexample of an orthopedic implant is a dental implant. Examples of dentalimplants include an artificial tooth root replacement, implant-supportedbridges and dentures. Other examples will be known to those of skill inthe art.

To be administered in an implant, as used herein, means that the fusionprotein may be associated with the implant, by for instance, adhesion,covalent or non-covalent bonding to the surface of the implant,entrapment in pores of a polymer coating of an implant, or mixing with acomponent of the implant, such as ceramic particles. If the ceramicparticles are porous, the fusion protein can be entrapped in the pores.By “entrapped in the pores” it is meant that diffusion of the fusionprotein out of the material is slowed due to the pore structure, notnecessarily that the fusion protein cannot diffuse out of the materialuntil the material breaks down.

For instance, the fusion protein can be entrapped in a biodegradablepolymer as described in U.S. Pat. No. 7,060,299. It may be formed intoparticles with a polysaccharide gum, and then the particles entrapped ina matrix of a polymer as described in U.S. Pat. No. 7,060,299. Thepolymer can be formed as a coating on the surface of an implant.

The fusion protein can also be bonded to a surface such as gold on animplant through sulfhydryls of the protein, as described in U.S. Pat.No. 6,428,579.

The fusion protein can be mixed with a ceramic or with ceramicparticles, including for example hydroxyapatite or tricalcium phosphate,both of which are often used as fillers for bone remodeling (U.S.Published Patent Application No. 20030091609).

A porous polymer can be formed by forming the polymer in an organicsolvent with particles of a material that is not soluble in the organicsolvent, such as salt or sugar crystals. After the polymer is cured, theparticles can be removed to expose the open pores by washing the polymermatrix in an aqueous solution that solubilizes the salt or sugarparticles. Incubating the polymer matrix with a solution of the fusionprotein can allow the fusion protein to diffuse into the pores of thepolymer and become entrapped therein (U.S. Published Patent ApplicationNo. 20030091609).

Other methods of adhering proteins to a surface of a material aredisclosed in U.S. Pat. No. 6,617,142. Still other methods are availableto those of skill in the art.

The fusion protein can be mixed with demineralized bone matrix (DBM).Demineralized bone matrices are prepared by acid extraction of allograftbone, resulting in loss of most of the mineralized component butretention of collagen and noncollagenous proteins, including growthfactors. DBM is used as a bone-graft substitute or extender. Since DBMcontains extensive amounts of collagen, the fusion proteins will bind tothe collagen of DBM if mixed with DBM in binding buffer.

In specific embodiments, the orthopedic implant includes hydroxyapatite,tricalcium phosphate, or demineralized bone matrix. In otherembodiments, the orthopedic implant includes a polymer. Many natural andsynthetic polymers may be included in an orthopedic implant (e.g., as acoating). Examples of natural porous polymers include gelatin, fibrin,collagen, elastin, hyaluronic acid, chondroitin sulfate, dermatansulfate, heparin sulfate, heparin, cellulose, chitin, chitosan, mixturesor copolymers thereof, or a wide variety of others typically disclosedas being useful in implantable medical devices. Examples of syntheticporous polymers include silicone, polyurethane, polysulfone,polyethylene, polypropylene, polyamide, polyester, polycarboxylic acids,polyvinylpyrrolidone (PVP), maleic anhydride polymers, polyamides,polyvinyl alcohols (PVA), polyethylene oxides, polyacrylic acidpolymers, polytetrafluoroethylene, polyhydroxyethylmethacrylic acid(pHEMA), polyaminopropylmethacrylamide (pAPMA),polyacrylamido-2-methylpropanesulf-onic acid (pAMPS), polyacrylamide,polyacrylic acid, mixtures or copolymers thereof, or a wide variety ofothers typically disclosed as being useful in implantable medicaldevices. Additional examples of synthetic porous polymers includebiodegradable synthetic porous polymers, such as polyglycolic acid,polylactic acid, polydiaxonone, poly(,-caprolactone), polyanhydrides,poly(3-hydroxybutyrate), poly(ortho esters), poly(amino acids),polyiminocarbonates, and mixtures or copolymers thereof.

Thus, another embodiment provides a method of promoting tissue growtharound an implant in a mammal comprising: administering to the mammal afusion protein comprising: (a) a collagen-binding polypeptide segment;linked to (b) a PTH/PTHrP receptor agonist polypeptide segment. Before,during, or after the step of administering the fusion protein, themammal receives an implant placed in contact with tissue in the mammal;and the step of administering the fusion protein is effective to promotetissue growth around the implant. The tissue growth promoted around theimplant may be bone, cartilage, or other tissue. In one embodiment, itmay be skin.

In a particular embodiment, the step of administering the fusion proteincomprises placing an implant in contact with tissue in the mammal,wherein the implant comprises the fusion protein.

In a particular embodiment, the implant is a dental implant.

In another embodiment, the implant is a bone graft.

In other embodiments, the implant is an orthopedic bone void filler, anadjunct to bone fracture stabilization, an intramedullary fixationdevice, a joint augmentation/replacement device, a bone fixation plate,a screw, a tack, a clip, a staple, a nail, a pin, a rod, an anchor, ascrew augmentation device, or a cranial reconstruction device.

In specific embodiments, the implant comprises intact bone. Here, in oneembodiment, the implant is incubated with the fusion protein for a timesufficient to allow the fusion protein to bind to collagen in the intactbone before implanting the implant in the mammal.

In specific embodiments, the implant comprises bone cement,hydroxyapatite, or demineralized bone.

In specific embodiments, the implant comprises osteoblasts.

In specific embodiments, the implant is predominantly plastic, metal, orceramic (i.e., the majority of its mass is plastic, metal, or ceramicmaterial).

Another embodiment provides a method of promoting hair growth in amammal comprising: administering to the mammal a fusion proteincomprising: a collagen-binding polypeptide segment; linked to aPTH/PTHrP receptor agonist polypeptide segment.

We have found that fusion proteins containing the receptor agonists weremore effective than those containing receptor antagonists in promotinghair growth in mice treated with cyclophosphamide to inducechemotherapy-induced alopecia (Example 8 below). A fusion proteincontaining a PTH/PTHrP receptor antagonist was also tested and alsoinduced some hair growth, but the hair that grew appeared less thick(data not shown). Thus, fusion proteins containing either a PTH/PTHrPreceptor agonist or antagonist can be used to promote hair growth, butfusion proteins containing a receptor agonist are preferred forchemotherapy-induced alopecia.

To promote hair growth, the fusion proteins may be administered locallyat a desired site of hair growth, e.g., by subcutaneous or intradermalinjection. The fusion proteins will bind to collagen in the skin nearthe site of subcutaneous or intradermal injection and remain bound atthe site for long-lasting effect. The fusion proteins can also beadministered systemically to promote hair growth. This is preferred totreat chemotherapy-induced alopecia.

In one embodiment of the method of promoting hair growth, the mammal isafflicted with chemotherapy-induced alopecia.

Another embodiment provides a method of promoting immune reconstitutionin a mammal comprising: administering to the mammal a fusion proteincomprising: (a) a collagen-binding polypeptide segment; linked to (b) aPTH/PTHrP receptor agonist polypeptide segment; wherein before, during,or after administering the fusion protein, the mammal receives anadministration of bone marrow stem cells. As used here, the term “bonemarrow stem cells” may refer to any stem cells that can implant in bonemarrow and differentiate into a variety of types of lymphocytes. Thus,the stem cells may be obtained, for instance, from umbilical cord blood,embryos, the mammal's own blood or bone marrow, or another mammal'sblood or bone marrow. Administration of the fusion protein is expectedto show an increase in survival following bone marrow ablation and astem cell transplant in mice. It is also expected to increase the rateof neutrophil number increase—i.e., neutrophil numbers are greater atspecific time points (e.g., 7, 14, 21, or 30 days) after transplant inpatients or experimental animals receiving the fusion protein inconjunction with the stem cell transplant than in a comparison group notreceiving the fusion protein.

In one embodiment, the stem cells will be umbilical cord blood stemcells. Umbilical cord blood is an especially useful alternative forpatients in need of a stem cell transplant who do not have anMHC-matched related or unrelated donor. But the number of stem cells ina single unit of umbilical cord blood is often insufficient forsuccessful engraftment after a bone marrow stem cell transplant (10).Administration of the fusion protein disclosed herein containing aPTH/PTHrP receptor agonist is expected to improve grafting of the stemcells and increase the odds of a successful graft with one or two unitsof umbilical cord blood.

In another embodiment, the stem cells will be autologous blood stemcells. Often too few stem cells are mobilized from a patient to supportautologous stem cell transplant. Administering the fusion protein isexpected to enhance the chance of successful engraftment when the numberof stem cells transplanted is less than optimal. It also is expected toenhance the chance of successful engraftment when the number of stemcells transplanted is considered adequate.

Preferably the fusion protein would be administered before or togetherwith administration of the stem cells to promote engraftment of stemcells in the bone marrow. But it may also be administered afteradministration of the stem cells.

Another embodiment provides a method of promoting bone marrow stem cellmobilization in a mammal comprising: administering to the mammal afusion protein comprising: (a) a collagen-binding polypeptide segment;linked to (b) a PTH/PTHrP receptor agonist polypeptide segment.Administering the fusion protein is expected to increase the number ofstem cells in circulating blood of the mammal (e.g., 7, 14, or 30 daysafter administering the fusion protein). In a specific embodiment, thismethod further comprises collecting stem cells from blood of the mammalafter the step of administering the fusion protein to the mammal.

Autologous stem cell transplantation cures lymphomas in many patientsand improves survival in multiple myeloma. But approximately 20% ofpatients do not mobilize sufficient stem cells to safely supportautologous stem cell transplantation (11). The fusion protein describedherein containing a PTH/PTHrP receptor agonist is expected to promotestem cell mobilization.

Another embodiment is expected to provide a method of treatingmyocardial infarction in a mammal comprising: administering to a mammalafter the mammal suffers a myocardial infarction a fusion proteincomprising: (a) a collagen-binding polypeptide segment; linked to (b) aPTH/PTHrP receptor agonist polypeptide segment.

Another embodiment provides a method of treating or preventing renalosteodystrophy in a mammal comprising: administering to the mammal afusion protein comprising: (a) a collagen-binding polypeptide segment;linked to (b) a PTH/PTHrP receptor antagonist polypeptide segment;wherein the mammal is afflicted with renal osteodystrophy or renaldisease. In this embodiment, the fusion protein is expected to beeffective to reduce bone loss in the mammal.

One embodiment is expected to provide a method of treating or reducingincidence of bone metastasis of cancer in a mammal comprising:administering to the mammal a fusion protein comprising: (a) acollagen-binding polypeptide segment; linked to (b) a PTH/PTHrP receptorantagonist polypeptide segment.

PTHrP is positively associated with bone metastasis (15, 16, 17). Breastcarcinoma metastatic to bone expresses PTHrP in more than 90% of cases,compared with 17% in metastases to nonbone sites (15). In a mouse model,human tumor cells transfected with a cDNA to overexpress human PTHrP hadincreased metastasis to bone (15). Conversely, administration of ananti-PTHrP antibody decreased bone metastases (15, 17).

Binding of PTHrP to its receptor alters the microenvironment of bonefavorably to promote metastasis. A fusion protein containing a CBDsegment and a PTH/PTHrP receptor antagonist will likely occupy thereceptor in bone and thus decrease the occurrence of metastasis. It isexpected to slow the growth of metastic tumors in bone.

In all the embodiments described herein, fusion proteins comprising (a)a collagen-binding polypeptide segment linked to (b) a PTH/PTHrPreceptor agonist polypeptide segment can be replaced by pharmaceuticalagents comprising (a) a collagen-binding polypeptide segment linked to(b) a PTH/PTHrP receptor agonist or a non-peptidyl PTH/PTHrP receptoragonist. An example of a non-peptidyl PTH/PTHrP receptor agonist iscompound AH3960 (19).

AH3960 contains two amino groups. These can be used to cross-link thecompound to amino groups on the collagen-binding polypeptide segmentthrough a cross-linker such as DSG (disuccinimidyl glutarate) or throughthe combination of SANH (succinimidyl-4-hydrazinonicotinate acetonehydrazone) and SFB (succinimidyl-4-formyl benzoate). AH3960 can becross-linked through its amino group to a carboxyl group of thecollagen-binding polypeptide segment by EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride). Theseproducts are available from Pierce (piercenet.com, Thermo FisherScientific Inc., Rockford, Ill.). Protocols and reaction conditions arealso available in the product literature from Pierce (piercenet.com).

Likewise, in the embodiments described herein involving receptorantagonist fusion proteins, fusion proteins comprising (a) acollagen-binding polypeptide segment linked to (b) a PTH/PTHrP receptorantagonist polypeptide segment can be replaced by pharmaceutical agentscomprising (a) a collagen-binding polypeptide segment linked to (b) aPTH/PTHrP receptor antagonist or a non-peptidyl PTH/PTHrP receptorantagonist.

Thus, another embodiment provides a pharmaceutical agent comprising: (a)a collagen-binding polypeptide segment linked to (b) a PTH/PTHrPreceptor antagonist, where the antagonist may be non-peptidyl.Non-peptidyl antagonists of the PTH/PTHrP receptor include compoundsdisclosed in (20), including compound 2 below:

Compound 2 can be coupled through its amino group to amino or carboxylgroups of the collagen-binding polypeptide segment as described abovefor compound AH3960. In compound 3 of reference (20), the amino group ofcompound 2 is replaced with a carboxyl group. This can be coupled toamino groups of the collagen-binding polypeptide segment with EDC.

In another embodiment of the pharmaceutical agents comprising (a) acollagen-binding polypeptide segment; linked to (b) a PTH/PTHrP receptoragonist polypeptide segment or antagonist polypeptide segment, segment(a) and segment (b) are separate polypeptides, and the two polypeptidesare linked by chemical cross-linking. The two polypeptides can becross-linked through amino groups by reagents including DSG(disuccinimidyl glutarate) or glutaraldehyde. They can also becross-linked through amino groups by derivatizing one polypeptide withSANH (succinimidyl-4-hydrazinonicotinate acetone hydrazone) and theother with SFB (succinimidyl-4-formyl benzoate), and then mixing the twoderivatized polypeptides to cross-link. The two polypeptides can becross-linked between an amino group of one polypeptide and a carboxyl ofthe other by reaction with EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride). Thepolypeptides can also be cross-linked (e.g., covalently coupled) by anyother suitable method known to a person of ordinary skill in the art.These cross-linking reagents are available from Pierce (piercenet.com,Thermo Fisher Scientific Inc., Rockford, Ill.). Protocols and reactionconditions are also available in the product literature from Pierce(piercenet.com). These and other applicable cross-linking methods aredescribed in U.S. published patent applications 20060258569 and20070224119.

Based on the data herein, the individual doses of pharmaceutical agentscomprising a collagen-binding polypeptide segment linked to a PTH/PTHrPreceptor agonist polypeptide segment can be approximately the same on amolar basis as doses used for PTH(1-34). But the pharmaceutical agentscomprising a collagen-binding polypeptide segment linked to a PTH/PTHrPreceptor agonist polypeptide segment can be administered lessfrequently, because linking the agonist to the collagen-bindingpolypeptide segment gives it much more prolonged activity in vivo.

The following examples are presented to illustrate various aspects ofthe disclosure without limiting the scope thereof.

EXAMPLES Example 1 Expression of PTH-Collagen-Binding Domain FusionProteins

A plasmid expressing a PTH-CBD fusion protein was constructed byinserting the PTH-CBD coding sequence into pGEX-5X-1 (GE Lifesciences).The sequence of the resulting plasmid is SEQ ID NO:3. Nucleotides 258 to1409 of SEQ ID NO:3 encode a fusion protein containingglutathione-S-transferase (GST) fused at its C terminus to a PTH-CBDfusion protein. SEQ ID NO:4 is the full encoded GST-PTH-CBD fusionprotein. Residues 222-225 are IEGR (SEQ ID NO:5), a factor Xa proteaserecognition site. Residues 226-383 of SEQ ID NO:4 correspond to SEQ IDNO:1 and are the PTH-CBD fusion protein. Factor Xa cleaves after the Argthat is amino acid residue 225 of SEQ ID NO:4 to release SEQ ID NO:1,the PTH-CBD fusion protein. Residues 1-33 of SEQ ID NO:1 are theN-terminal 33 residues of PTH. Residues 38-158 are a collagen-bindingdomain (CBD) of the ColH collagenase of Clostridium histolyticum. TheCBD of the fusion protein corresponds to residues 901-1021 of ColH (SEQID NO:6). Residues 34-37 of SEQ ID NO:1 are a linker segment.

A second PTH-CBD fusion protein, PTH-PKD-CBD (SEQ ID NO:2), wasexpressed from the a plasmid otherwise identical to SEQ ID NO:3 with alonger insert segment from the colH gene to express. Like SEQ ID NO:1,it was expressed as part of a GST fusion protein and cleaved from GST byFactor Xa. Residues 1-33 of SEQ ID NO:2 are the N-terminal 33 residuesof PTH. Residues 34-36 are a linker segment. And residues 37-251 areresidues 807-1021 of ColH. This fusion protein includes a polycystickidney disease (PKD) domain of ColH (residues 807-900 of ColH), inaddition to the collagen binding domain of residues 901-1021 of ColHfound in both SEQ ID NO:1 and SEQ ID NO:2. It was thought that includingthe PKD domain might minimize domain-domain interferences or othersteric hindrances between the PTH domain and CBD domain.

Purification of CBD Fusion Proteins—

E. coli BL21 was transformed with the recombinant plasmids. Each clonewas grown in one liter of 2YT-G medium to an optical density at 600 nmof 0.7. Isopropyl-1-thio-beta-D-galactopyranoside was added to a finalconcentration of 0.1 mM, and cells were grown for a further 2 hours. Inorder to prevent proteolyis during the purification procedures,phenylmethylsulfonylfluoride was added to the culture to a finalconcentration of 1 mM. Cells were harvested by centrifugation, anddisrupted in a French pressure cell. Cell debris was removed bycentrifugation, and the cleared lysate was used for the purification ofthe fusion protein by a batch method using glutathione-SEPHAROSE 4Bbeads (volume, 4-ml; GE Lifesciences) as described by the manufacturer.The GST-tag of each fusion protein was cleaved by incubation with FactorXa (New England Biolabs, 0.2 μg/mg of fusion protein) for 20 h at roomtemperature. The cleaved protein fractions were dialyzed three timesagainst 1 liter of 50 mM Tris-HCl (pH7.5), 100 mM NaCl at 4° C. toremove glutathione. The N-terminal GST fragment was removed by applyingthe fraction to a glutathione-SEPHAROSE 4B column (bed volume, 2 ml).Ten amino acid residues from the N terminus were confirmed for eachfragment on an automatic protein sequencer (Model 492, Perkin-Elmer).The molecular mass of the purified C-terminal fragment was confirmed bymatrix-assisted laser desorption time-of-flight mass spectrometry(MALDI-TOF MS).

Example 2 Demonstration of Collagen Binding by the PTH-CBD FusionProteins

Five mg insoluble collagen type I, (C-9879; Sigma) was added to an ULTRAFREE micro centrifugal device, 0.22 micrometer low-binding DURAPOREmembrane (Millipore, Bedford, Mass.) and placed in a micro centrifugetube (Catalogue No:UFC30GV00-Millipore). All steps were carried at roomtemperature unless otherwise specified. Collagen binding buffer (200microliters) (50 mM Tris-HCl, pH 7.5, 5 mM CaCl₂) was added to swell thecollagen fibers. After incubation for 30 minutes, the tube wascentrifuged at 15,000 g for 15 minutes. Centrifugation was repeatedafter changing the direction of the tube in the rotor. The collagenprecipitate was resuspended in 60 mcl of collagen binding buffercontaining 100 pmole of fusion protein and incubated for 30 minutes. Themixture was then centrifuged through the device at 15,000×g for 15minutes. Proteins bound to the collagen would be retained by the filteralong with the collagen. Proteins that do not bind to collagen wouldpass through in the filtrate. The filtrate was analyzed by SDS-PAGE.

FIG. 1 shows a photograph of the SDS-PAGE gel. Lane 1 on the left ismolecular weight markers. Lane 2 is the filtrate of a mixture containingPTH-PKD-CBD fusion protein filtered without collagen. Lane 3 shows thefiltrate of a mixture of PTH-PKD-CBD fusion protein with collagen. Lanes4 and 5 show the filtrate of the PTH-CBD fusion protein incubutedwithout and with collagen, respectively. The result shows that bothfusion proteins failed to pass through the filter when incubated withcollagen, but did pass through when incubated without collagen. Thisshows both fusion proteins bound to collagen.

Example 3 In Vitro Biological Activity of PTH-CBD Fusion Proteins

HKrK-B7 cells, which are LLCPK cells stably transfected with the humanPTH1R, were kindly provided by Tom Gardella, Endocrine Unit,Massachusetts General Hospital. The cells are described in reference(7). HKrK-B7 cells were grown in 24 well plates to 90 percentconfluence, which was typically achieved 2-3 days after initial seeding.The culture media was DMEM (with L-glutamine)+10% fetal bovine serum(FBS).

When the cells reached 90% confluence, the cells were rinsed once with0.5 ml binding buffer (50 mM Tris-HCl, pH 7.8, 100 mM NaCl, 2 mM CaCl₂,5 mM KCl, 0.25% horse serum, 0.0025% fetal bovine serum). The plate wasplaced on ice, and 200 microliters IBMX buffer (DMEM without antibioticand FBS, 35 mM HEPES, pH 7.4, 3-isobutyl-1-methylxanthine (IBMX), 1mg/ml bovine serum albumin) was added per well. IBMX is aphosphodiesterase inhibitor. Peptide or PTH was added at the indicatedconcentrations in 100 micoliters binding buffer. The cells were thenincubated with the peptide, PTH, or no addition (control) for 1 hour atroom temperature. The media was then removed and the plates were placedon dry ice to freeze the cells for 3 minutes. 500 microliters 50 mM HClwas next added to each well. The plates were kept frozen until theimmunoassay.

cAMP concentration was measured by immunoassay (Biomedical Technologies,Inc., Stoughton, Mass., USA; cAMP EIA kit, #BT-730).

The results of the cAMP concentration from the lysed cells in the wellsis shown in FIG. 2 for cells incubated with from 1×10⁻¹² M to 1×10⁻⁷ Mfusion peptide or PTH(1-34). PTH(1-34), PTH-CBD (SEQ ID NO:1), andPTH-PKD-CBD (SEQ ID NO:2) all stimulated cAMP synthesis to a similarextent.

Example 4 In Vivo Activity of PTH-CBD Fusion Proteins

Healthy female C57BL/6J mice, 5-8 weeks age and 13-18 grams, werepurchased from the Jackson laboratory (Bar Harbor, Me., USA) and theywere housed in cages at the Animal facility in Ochsner Clinic Foundationunder standard conditions. Animals were maintained for a 2-weekacclimation period prior to experiments.

Baseline whole body DEXA (dual emission x-ray absorptiometry)measurements were obtained in duplicate for each animal using a HologicQDR-1000plus instrument adapted for application in the mouse as follows.An ultrahigh resolution mode (line spacing 0.03950 cm and resolution0.03749 cm) was used. The animals were anesthetized with pentobarbitaland positioned in the prone position for DEXA scanning. Bone mineraldensity (BMD) was determined within an 8×16 pixel box covering theregion of the lumbar spine. BMD for each single pixel vertical stripewas measured, and the peak values were determined. Validity for thistechnique was ascertained by comparing the duplicate measurements ineach mouse.

Animals were injected intraperitoneally weekly for eight weeks witheither vehicle alone (collagen binding buffer, pH 7.5, 50 mM Tris HCl, 5mM CaCl₂) or vehicle containing PTH analogs as follows:

Group A (8 animals): vehicle

Group B (6 animals): 80 μg/kg/dose of human PTH(1-34)

Group C (6 animals): 546 μg/kg/dose of PTH-PKD-CBD (SEQ ID NO:2)

Group D (6 animals): 344 μg/kg/dose of PTH-CBD (SEQ ID NO:1)

The doses of the three PTH compounds were adjusted based on theirmolecular weights, such that each was given at the same molar equivalent(0.02 micromoles/kg/dose).

One week after the 8^(th) injection, animals were sacrificed with alethal dose of pentobarbital. Duplicate BMD measurements were obtainedfor each mouse by the technique described above. Percent increase in BMDfor each mouse was calculated, and the results (average+/−standarderror) are shown in FIG. 3. Statistical significance was determinedusing a one-tailed paired T test. Statistically significant differencesfrom vehicle control are shown by * (p<0.05) and ** (p<0.01) in FIGS. 3and 4.

At the conclusion of the study, lumbar spine segments of the mice werealso excised from the soft tissue and BMD measurements of the excisedspine segments were taken. The BMD results of the excised spine segmentsare the average for the entire bone segment, not peak BMD measurementslike those that were obtained from the whole animal scans.

The statistical comparisons used were ANOVA across groups (p<0.05), andBonferroni comparisons of each group vs. control.

The PTH-CBD fusion protein (SEQ ID NO:1) produced an average 17%increase in BMD over the 8-week treatment period. Both PTH(1-34) and thePTH-PKD-CBD fusion protein (SEQ ID NO:2) produced approximately a 7.5%increase in bone mineral density. The mice in the vehicle control grouphad a 5% increase in BMD over the 8-week treatment period. (FIG. 3.)Both PTH-CBD (p<0.01) and PTH-PKD-CBD (p<0.05) fusion proteins producedBMD increases that were statistically significantly greater than vehiclecontrols, while PTH(1-34) did not. But the PTH-CBD fusion gaveapproximately twice the BMD increase of both PTH(1-34) and thePTH-PKD-CBD fusion protein. (FIG. 3)

The BMD of excised lumbar spine segments of the four groups of mice atthe conclusion of the 8-week treatment period are shown in FIG. 4.Again, the PTH-CBD group was statistically significantly different fromthe vehicle control (p<0.05). Differences between other groups withvehicle control and with each other did not reach statisticalsignificance.

Serum calcium levels were also measured in the mice before, during, andafter the study. PTH with daily injection is known to carry a risk ofhypercalcemia. There was no difference in serum calcium levels betweenany of the groups, indicating that the PTH-CBD fusion proteins did notcause hypercalcemia (FIG. 5).

Serum alkaline phosphatase levels were also measured. Serum alkalinephosphatase was increased in the PTH(1-34), PTH-PKD-CBD, and PTH-CBDgroups (FIG. 6). Elevated alkaline phosphatase is correlated withhyperparathyroidism and periods of bone growth. Thus, this is evidenceof increased bone turnover with all three agents.

Staining of tibial sections with hematoxylin and eosin showed increasedtrabecular and cortical bone in mice treated with 8 weeks of PTH-CBDversus vehicle control (FIG. 7).

No evidence of bone tumors in mice in any of the groups was found byDEXA or post-mortem examination.

We conclude that the PTH-CBD fusion protein is more active thanPTH(1-34) in promoting bone mineral density increase in vivo.

Example 5 Monthly Administration of PTH-CBD In Vivo

With the encouraging results showing efficacy of PTH-CBD to increasebone mineral density after weekly administration, we next tested theefficacy of this fusion protein with monthly administration. Micereceived intraperitoneal injection of PTH-CBD (344 μg/kg/dose), PTH (80μg/kg/dose), or vehicle alone monthly in buffer as described in Example4. There were 10 mice in each group. Bone mineral density (BMD) wasmeasured by DEXA as described in Example 4 every 2 months. DEXAmeasurements were correlated to absolute bone mineral density bycorrelation between DEXA measurements and measurements from excisedtissue in the weekly study of Example 4.

Serial measurements of BMD every 2 months showed that monthlyadministration of PTH-CBD resulted in significant increases in BMD after4 months of therapy, which were sustained for 6 months of therapy (FIG.8) (p<0.01, shown by ** in FIG. 8). Not surprisingly, monthlyadministration of PTH(1-34) had no effect on bone mineral density. After6 months (as indicated by the arrow in FIG. 8), we discontinuedadministration of PTH-CBD, and subjected the animals in the PTH(1-34)group to 2 weeks of daily therapy. Measurement of BMD 2 months latershowed that the gains in bone mineral density after PTH-CBDadministration were sustained (despite the decline in BMD in the vehiclecontrol group, expected for age), and that the daily administration ofPTH(1-34) resulted in increases in BMD which approached but did notreach those of the PTH-CBD group.

The mice were then followed for another 6 months, and the data showedthat the BMD of the PTH(1-34) and PTH-CBD groups declined in paralleland remained higher than the untreated vehicle control mice.

Serum concentration of alkalaline phosphatase was also measured in thesegroups of mice at the 48-week time point. The results are shown in FIG.10. Even at 48 weeks, 22 weeks after the last administration of thePTH-CBD fusion protein, alkaline phosphatase concentration was elevatedin the group receiving the PTH-CBD fusion protein compared to thevehicle control mice and mice that received PTH(1-34).

Conclusion

Together with the data in Example 4, these data indicate that monthlyadministration of PTH-CBD showed at least equal efficacy to dailyinjection of PTH in promoting an increase in bone mineral density.Importantly, the dose of PTH-CBD given in each injection is the molarequivalent of the daily dose of PTH(1-34); thus, the total administereddose is actually 1/30 of the dose with PTH(1-34). The data suggests thateven longer dosing intervals than monthly may be effective, and that theeffects on BMD are sustained for a longer time after cessation oftherapy with PTH-CBD than with PTH(1-34).

Example 6 3- and 6-Monthly Administration of PTH-CBD In Vivo

With the encouraging results showing efficacy of PTH-CBD to increasebone mineral density after monthly administration, we next tested theefficacy of this fusion protein with administration every 3 or every 6months. Mice received intraperitoneal injection of PTH-CBD (344μg/kg/dose×1) (CBD-PTH-6 of FIG. 9), PTH-CBD (344 μg/kg/dose at 0 and 3months) (CBD-PTH-6 of FIG. 9), PTH(1-34) (80 μg/kg/dose daily for 2weeks), or vehicle alone (xl) in buffer as described in Example 4. Therewere eleven mice in each group. Bone mineral density (BMD) was measuredby DEXA at 3 month and monthly thereafter. The study is ongoing, anddata are available up to the 5 month time point

Serial measurements of BMD showed that a single dose of PTH-CBD resultedin significant increases in BMD after 4 months of therapy (FIG. 9).Administration of the second dose of PTH-CBD at the 3 month time pointdid not cause further increases in BMD at the 4 and 5 month time points.Daily administration of PTH(1-34) for 2 weeks caused the expectedincrease in BMD at 3 months, but by 5 months the BMD had declined backto control levels. The mice in this study will be followed for anadditional 7 months.

Conclusion

Together with the data in Examples 4 and 5, these data suggest that asingle dose of PTH-CBD is sufficient to promote sustained increases inbone mineral density. Importantly, the dose of PTH-CBD given in eachinjection is the molar equivalent of the daily dose of PTH(1-34); thus,the total administered dose is actually 1/14 of the dose of PTH(1-34)over the 5 month interval for which we have data at this time. We willcontinue to collect data on this study for another 7 months. The dataalso indicate that the effects on BMD are sustained for a longer timeafter cessation of therapy with PTH-CBD than with PTH(1-34).

Example 7 Preliminary Dose and Time Response Study

To determine roughly the optimal dose of PTH-CBD, a single dose of thefusion protein was given by subcutaneous administration to mice at arange of doses from 2 to 8,000 micrograms/kg and the BMD of the mice wastested by DEXA every 4 weeks for 20 weeks. At the highest dose, the BMDdecreased between 4 weeks and 12 weeks and then increased. It thusappeared to have a transient catabolic effect and then a possibleanabolic effect. Intermediate doses of 40-400 micrograms/kg, which spansthe dose of 344 micrograms/kg used in Example 4 and 5, appeared to havethe greatest anabolic effect over the first 8 weeks. The lowest dosetested, 2 micrograms/kg appeared to have less anabolic effect over thefirst 16 weeks. (FIG. 11)

Example 8 Use of PHT-CBD to Promote Hair Growth

There are reports that PTH agonists and antagonists can modulate hairgrowth in animal models of genetic hair loss and after administration ofchemotherapy (8,9). We tested whether PTH-CBD could, after subcutaneousadministration, alter the pattern of hair growth afterchemotherapy-induced hair loss with cyclophosphamide.

Materials and Methods:

Healthy female C57BL/6J mice (as in Example 4) were treated with 150mg/kg cyclophosphamide every month for 3 months. The chemotherapeuticagent caused hair thinning and color change from black to white. Weadditionally shaved a spot on the back. At the spot of hair removal, weinjected PTH-CBD subcutaneously at a dose of 320 mg/kg. We also testedinjection of a CBD fusion protein containing a PTH/PTHrP receptorantagonist (SEQ ID NO:9). This fusion protein was made by inserting athrombin cleavage sequence (Leu-Val-Pro-Arg-Gly-Ser, SEQ ID NO:12)between the GST and PTH(1-33) segments of the fusion protein of SEQ IDNO:1. The resultant GST-PTH-CBD fusion protein is cleaved by thrombinbetween the Arg and Gly residues of the thrombin cleavage sequence torelease the Gly-Ser-PTH-CBD fusion protein of SEQ ID NO:9.

Results:

The PTH-CBD treated animals showed more rapid regrowth of hair at thespot of removal, and the chemotherapy-induced thinning and color changeof the hair were both reversed, even at sites distant from the PTH-CBDinjection site (FIG. 12). A CBD fusion protein containing a PTH/PTHrPreceptor antagonist was also tested in pilot studies. But the antagonistfusion protein produced only peach fuzz hair at the site of injectionand did not work as well as the PTH-CBD agonist fusion protein (resultsnot shown). The antagonist fusion protein produced more hair thanvehicle control treatment (results not shown).

Conclusion

PTH-CBD can reverse chemotherapy-induced alopecia, and the effects arenot restricted to the site of injection.

Example 9 Use of PHT-CBD to Promote Immune Reconstitution

Female C57Bl/6 mice are irradiated with 10 Gy of radiation (¹³⁷Cssource). 24 hours later, mice are injected with 2×10⁵ bone marrowmononuclear cells (BMMNC) from a donor B6. SJL mouse. Immediately beforereceiving the BMMNC, the recipient mice are also injected with saline(vehicle control), 344:g/kg PTH-CBD (SEQ ID NO:1), or 80:g/kg PTH(1-34).

A portion or all of the mice receiving BMMNC alone are expected to die.A greater percentage of mice receiving PTH(1-34) are expected tosurvive. A still greater percentage of mice receiving PTH-CBD areexpected to survive.

It is also expected that neutrophil count will increase faster in micereceiving the PTH-CBD fusion than in mice receiving an equimolar amountof PTH or receiving vehicle control.

Example 10 Use of PTH-CBD to Promote Bone Marrow Stem Cell Mobilization

Six- to 8-week old male C57BL/6 mice are injected subcutaneously with asingle dose of 80 mcg/kg PTH(1-34) or 344 mcg/kg PTH-CBD (SEQ ID NO:1)or saline (vehicle control). Fourteen days later, peripheral blood iscollected from the mice, and c-KIT/Sca-1 cells are determined byfluorescence activated cell sorting (FACS) (21). It is determined thatPTH-CBD causes a greater increase in c-KIT/Sca-1 double positive cellsthan a single dose of PTH(1-34).

To test the ability of stem cells mobilized with PTH-CBD to repopulate,blood is collected 14 days after treatment with PTH, PTH-CBD, or vehiclecontrol as described above. Red cells are lysed as described in (22).Total collected cells from 900 mcl of blood is transfused into a mousethat was subjected to a lethal dose of radiation (900 cGy) 24 hoursbefore. A larger percentage of recipient mice are expected to survivewhen given blood cells from a donor mouse treated with PTH-CBD than froma mouse treated with PTH(1-34) or vehicle control. Further, it isexpected that administering the fusion protein will increase the numberof stem cells in circulating blood of the mammal (e.g., 7, 14, or 30days after administering the fusion protein)

Example 11 Use of a CBD-PTH/PTHrP Receptor Antagonist Fusion Protein forthe Prevention and Treatment of Bone Metastasis of Breast Cancer

When administered as a daily injection, PTH(1-34) stimulates bone growthin various species and in osteoporotic women. However, continuousadministration of PTH as an infusion (i.e. parathyroid adenoma) resultsin bone loss.

Breast cancer metastasizes to bone by producing a factor, PTH-relatedpeptide (PTHrP), which activates the PTH/PTHrP receptor, increasing boneturnover in the local region. The removal of bone tissues which resultsfrom this cascade creates a void in the bone where cancer cells can growand causes release of growth factors from the remodeled collagen matrixwhich promote tumor growth. In this study, we show that a PTH-CBDantagonist peptide has the ability to treat or prevent (reduce incidenceof) bone metastasis of breast cancer. The model used is theimmunodeficient nude mouse.

Animals receive a single injection of MCF-7 human breast cancer cellstagged with a phosphorescent probe. Animals are imaged weekly using awhole body imager to assess for bone metastatic lesions. Once 2 or morelesion are present in each animal, the animals receive a singleinjection of PTH(7-33)-CBD or vehicle control. Weekly imaging iscontinued for an additional 2 months to monitor growth of existingmetastases and appearance of new metastases.

Experimental Methods

22 Nude mice, aged 3-5 weeks and 13-18 grams are obtained. Initialweight of the animals is recorded along with their general healthcondition. Animals are maintained for a 2 week acclimation period priorto experiments. (final age 5-8 weeks).

Baseline images are obtained from each animal using theBioluminescent/Fluorescent Imager (Xenogen Biosciences, Cranbury, N.J.)whole body imager after isoflourane anesthesia. Animals then receive asingle injection of MCF-7 cells stably transfected with a plasmidexpressing firefly luciferase (23, 24). Animals are re-imaged followingthe injection and on a weekly basis thereafter to monitor for bonemetastasis.

When 2 or more metastatic lesions are presenting the bones of eachmouse, the animals will be divided randomly into 2 groups:

Group 1: 11 animals—is administered with vehicle intraperitoneally once.Group 2: 11 animals—is administered with 344 mcg/kg of PTH(7-33)-CBD(SEQ ID NO:10) intraperitoneally once.

Animals are sedated with isoflourane and whole body images are obtainedon a weekly basis for a 2 month period.

Data Analysis:

During the experimental period, animals are weighed and examined weeklyto detect any signs of illness. Whole body images are analyzed todetermine the number of metastatic lesions and intensity of theluminescent light emmission from each lesion.

At the end of the experimental period the animals will be sacrificed byinjecting a lethal dose of pentobarbital (100 mg/kg). Regions of thebone which contain(ed) metastatic lesions at any point during the studyare prepared for histological examination.

Results:

Mice injected with PTH(7-33)-CBD are expected to develop fewermetastatic bone lesions and have slower growth of metastatic bonelesions than mice receiving vehicle control.

Example 12 Use of a CBD-PTH/PTHrP Receptor Antagonist Fusion Protein forthe Prevention and Treatment of Renal Osteodystrophy

Renal osteodystrophy is a bone disease that occurs when kidneys fail tomaintain the proper levels of calcium and phosphorus in the blood. It'sa common problem in people with kidney disease and affects 90 percent ofdialysis patients. Renal osteodystrophy is a key cause of fractures inpatients with chronic kidney disease. In this study, we show thatPTH-CBD antagonist peptide has the ability to treat or preventosteodystrophy. The model used is normal female mice fed with a highphosphorus diet to induce renal osteodystrophy.

Animals then receive a single injection of PTH(7-33)-CBD or vehiclecontrol. Animals are maintained for 6 months after the initial dosingperiod to assess the duration of the therapeutic effects. Bone mineraldensity and alkaline phosphatase levels are measured on a monthly basis.

Experimental Plan:

Healthy female normal C57BL/67 mouse, aged 3-5 weeks and 13-18 grams areobtained. Initial weight of the animals is recorded along with theirgeneral health condition. Animals are maintained for a 2 weekacclimation period prior to experiments (final age 5-8 weeks).

Animals are fed with high phosphorus diet to induce renal osteodystrophy(ROD). The animals are checked periodically for their health status. Theblood samples are collected to assess the calcium, phosphorus, PTH andVitamin D levels. Renal osteodystrophy results from an abnormallyelevated serum phosphate (hyperphosphatemia) and low serum calcium(hypocalcemia), both of which are due to decreased excretion ofphosphate by the damaged kidney, low vitamin D levels or tertiaryhyperparathyroidism (dysfunction of the parathyroid gland due toconstant stimulation).

Baseline bone mineral density measurements are also be made.

The animals are divided into the following groups:

Group 1: 11 animals—are administered vehicle intraperitoneally once.Group 2: 11 animals—are administered with 344 mcg/kg of PTH(7-33)-CBD(SEQ ID NO:10) intraperitoneally once.

Animals are sedated with pentobarbital and bone mineral density (BMD) ismeasured at the start of the study and monthly for the duration of thestudy (6 months). Blood samples are obtained from tail clipping at thestart of the study and every month (under sedation as above).

Data Analysis:

During the experimental period, animals are weighed and examined weeklyto detect any signs of illness. Bone mineral density measurements areanalyzed by ANOVA at each time point. Alkaline phosphatase and calciumvalues are measured from each blood sample and analyzed by ANOVA at eachtime point.

At the end of the experimental period the animals are sacrificed byinjecting a lethal dose of pentobarbital (100 mg/kg). Blood samples arecollected to perform biochemical assays (intact PTH, calcium,phosphorus, alkaline phosphatase, osteocalcin). Quantitative bone assaysinclude histomorphometry, BMC and BMD of the total body and excisedspine, and assessment of biomechanical properties. Data is analyzed byANOVA.

Results:

The animals injected with PTH(7-33)-CBD are expected to respond withincreases or slower decreases in all measures of bone mineral density ascompared to mice receiving vehicle control. Mice injected withPTH(7-33)-CBD are expected also to show trabecular bone growth or slowerloss of trabecular bone than mice receiving vehicle control.

SEQUENCE LISTING SUMMARY

-   SEQ ID NO:1 PTH-CBD fusion protein-   SEQ ID NO:2 PTH-PKD-CBD fusion protein-   SEQ ID NO:3 vector expressing PTH-CBD fusion protein precursor.-   SEQ ID NO:4 GST-PTH-CBD fusion protein expressed by vector.-   SEQ ID NO:5 Factor Xa recognition sequence.-   SEQ ID NO:6 ColH collagenase.-   SEQ ID NO:7 PTH.-   SEQ ID NO:8 PTHrP.-   SEQ ID NO:9 CBD fusion protein with PTH receptor antagonist.-   SEQ ID NO:10 PTH(7-33)-CBD fusion protein-   SEQ ID NO:11 PTH/PTHrP antagonist Gly-Ser-PTH(1-33)-   SEQ ID NO:12 Thrombin recognition sequence.

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All patents, patent documents, and other references cited areincorporated by reference.

1.-60. (canceled)
 61. A composition comprising: a collagen-bindingpolypeptide segment covalently linked to a PTH/PTHrP receptor agonist;wherein the collagen-binding polypeptide segment is a bacterial collagenbinding polypeptide segment, wherein the PTH/PTHrP receptor agonistcomprises residues 1-14 of SEQ ID NO: 1, and wherein thecollagen-binding polypeptide segment comprises a polypeptide fragmentconsisting of at least 10 consecutive amino acids of residues 38-158 ofSEQ ID NO:
 1. 62. The composition of claim 61, wherein thecollagen-binding polypeptide segment and the PTH/PTHrP receptor agonistare chemically cross-linked to each other or are polypeptide portions ofa fusion protein.
 63. The composition of claim 61, wherein thecomposition has at least 50% greater activity than PTH(1-34) as measuredby increased bone mineral density after eight weeks of weeklyadministration of the composition to a subject in need thereof at equalmolar doses of the PTH.
 64. The composition of claim 61, wherein thePTH/PTHrP receptor agonist is a polypeptide and the N-terminus of thecollagen-binding polypeptide segment is linked directly or through alinker polypeptide segment to the C-terminus of the PTH/PTHrP receptoragonist polypeptide.
 65. The composition of claim 61, wherein thePTH/PTHrP receptor agonist comprises residues 1-33 of SEQ ID NO: 1, SEQID NO: 7, or residues 1-34 of SEQ ID NO:
 7. 66. The composition of claim61, wherein the composition further comprises residues 37-130 of SEQ IDNO: 2 covalently linked to the collagen-binding polypeptide segment andthe PTH/PTHrP receptor agonist.
 67. (canceled)
 68. A method of treatinga medical condition comprising administering an effective amount of thecomposition of claim 61 to a mammal in need of treatment for the medicalcondition, wherein the medical condition is selected from the groupconsisting of promoting bone growth, promoting hair growth, promotingtissue growth, promoting immune reconstitution, promoting bone marrowstem cell mobilization and treating myocardial infarction.
 69. Themethod of claim 68, wherein administering the composition to the mammalincreases trabecular bone mineral density or cortical bone mineraldensity or trabecular bone mineral volume or cortical bone mineralvolume.
 70. The method of claim 68, wherein the composition isadministered before, during or after administration of an implant or incombination with an implant.
 71. The method of claim 68, wherein theimplant is a dental implant or a bone graft.
 72. The method of claim 68,wherein the implant comprises intact bone, bone cement, hydroxyapatite,demineralized bone, osteoblasts or combinations thereof.
 73. The methodof claim 68, wherein the composition is administered by injection. 74.The method of claim 68, wherein the composition is administered inaqueous solution at pH below about 5.0.